Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors

ABSTRACT

The present invention provides a process for growing eukaryotic microorganisms which are capable of producing lipids, in particular lipids containing polyenoic fatty acids. The present invention also provides a process for producing eukaryotic microbial lipids.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/352,421, filed Feb. 10, 2006, which is a continuation ofU.S. patent application Ser. No. 10/371,394, filed Feb. 21, 2003, nowabandoned, which is a continuation of U.S. patent application Ser. No.09/771,352, filed Jan. 26, 2001, now U.S. Pat. No. 6,607,900, whichclaims the benefit of priority under 35 U.S.C. §119(e) from ProvisionalPatent Application Ser. No. 60/178,588, filed on Jan. 28, 2000. Each ofthe foregoing applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is directed to a novel process for growingmicroorganisms and recovering microbial lipids. In particular, thepresent invention is directed to producing microbial polyunsaturatedlipids.

BACKGROUND OF THE INVENTION

Production of polyenoic fatty acids (fatty acids containing 2 or moreunsaturated carbon-carbon bonds) in eukaryotic microorganisms isgenerally known to require the presence of molecular oxygen (i.e.,aerobic conditions). This is because it is believed that the cis doublebond formed in the fatty acids of all non-parasitic eukaryoticmicroorganisms involves a direct oxygen-dependent desaturation reaction(oxidative microbial desaturase systems). Other eukaryotic microbiallipids that are known to require molecular oxygen include fungal andplant sterols, oxycarotenoids (i.e., xanthophyls), ubiquinones, andcompounds made from any of these lipids (i.e., secondary metabolites).

Eukaryotic microbes (such as algae; fungi, including yeast; andprotists) have been demonstrated to be good producers of polyenoic fattyacids in fermentors. However, very high density cultivation (greaterthan about 100 g/L microbial biomass, especially at commercial scale)can lead to decreased polyenoic fatty acid contents and hence decreasedpolyenoic fatty acid productivity. This may be due in part to severalfactors including the difficulty of maintaining high dissolved oxygenlevels due to the high oxygen demand developed by the high concentrationof microbes in the fermentation broth. Methods to maintain higherdissolved oxygen level include increasing the aeration rate and/or usingpure oxygen instead of air for aeration and/or increasing the agitationrate in the fermentor. These solutions generally increase the cost oflipid production and can cause additional problems. For example,increased aeration can easily lead to severe foaming problems in thefermentor at high cell densities and increased mixing can lead tomicrobial cell breakage due to increased shear forces in thefermentation broth (this causes the lipids to be released in thefermentation broth where they can become oxidized and/or degraded byenzymes). Microbial cell breakage is an increased problem in cells thathave undergone nitrogen limitation or depletion to induce lipidformation, resulting in weaker cell walls.

As a result, when lipid producing eukaryotic microbes are grown at veryhigh cell concentrations, their lipids generally contain only very smallamounts of polyenoic fatty acids. For example, the yeast Lipomycesstarkeyi has been grown to a density of 153 g/L with resulting lipidconcentration of 83 g/L in 140 hours using alcohol as a carbon source.Yet the polyenoic fatty acid content of the yeast at concentrationgreater than 100 g/L averaged only 4.2% of total fatty acids (droppingfrom a high of 11.5% of total fatty acid at a cell density of 20-30g/L). Yamauchi et al., J. Ferment. Technol., 1983, 61, 275-280. Thisresults in a polyenoic fatty acid concentration of only about 3.5 g/Land a polyenoic fatty acid productivity of only about 0.025 g/L/hr.Additionally, the only polyenoic fatty acid reported in the yeast lipidswas C18:2.

Another yeast, Rhodotorula glutinus, has been demonstrated to have alipid productivity of about 0.49 g/L/hr, but also a low overallpolyenoic fatty acid content in its lipids (15.8% of total fatty acids,14.7% C18:2 and 1.2% C18:3) resulting in a polyenoic fatty acidproductivity in fed-batch culture of only about 0.047 g/L/hr and 0.077g/L/hr in continuous culture.

Present inventors have previously demonstrated that certain marinemicroalgae in the order Thraustochytriales can be excellent producers ofpolyenoic fatty acids in fermentors, especially when grown at lowsalinity levels and especially at very low chloride levels. Others havedescribed Thraustochyrids which exhibit a polyenoic fatty acid (DHA,C22:6n-3; and DPA, C22:5n-6) productivity of about 0.158 g/L/hr, whengrown to cell density of 59 g/L/hr in 120 hours. However, thisproductivity was only achieved at a salinity of about 50% seawater, aconcentration that would cause serious corrosion in conventionalstainless steel fermentors.

Costs of producing microbial lipids containing polyenoic fatty acids,and especially the highly unsaturated fatty acids, such as C18:4n-3,C20:4n-6, C20:5n3, C22:5n-3, C22:5n-6 and C22:6n-3, have remained highin part due to the limited densities to which the high polyenoic fattyacid containing eukaryotic microbes have been grown and the limitedoxygen availability both at these high cell concentrations and thehigher temperatures needed to achieve high productivity.

Therefore, there is a need for a process for growing microorganisms athigh concentration which still facilitates increased production oflipids containing polyenoic fatty acids.

SUMMARY OF THE INVENTION

The present invention provides a process for growing eukaryoticmicroorganisms which are capable of producing at least about 20% oftheir biomass as lipids and a method for producing the lipids.Preferably the lipids contain one or more polyenoic fatty acids. Theprocess comprises adding to a fermentation medium comprising eukaryoticmicroorganisms a carbon source, preferably a non-alcoholic carbonsource, and a nitrogen source. Preferably, the carbon source and thenitrogen source are added at a rate sufficient to increase the biomassdensity of the fermentation medium to at least about 100 g/L.

In one aspect of the present invention, the fermentation conditioncomprises a biomass density increasing stage and a lipid productionstage, wherein the biomass density increasing stage comprises adding thecarbon source and the nitrogen source, and the lipid production stagecomprises adding the carbon source without adding the nitrogen source toinduce nitrogen limiting conditions which induces lipid production.

In another aspect of the present invention, the amount of dissolvedoxygen present in the fermentation medium during the lipid productionstage is lower than the amount of dissolved oxygen present in thefermentation medium during the biomass density increasing stage.

In yet another aspect of the present invention, microorganisms areselected from the group consisting of algae, fungi, protists, andmixtures thereof, wherein the microorganisms are capable of producingpolyenoic fatty acids or other lipids which requires molecular oxygenfor their synthesis. A particularly useful microorganisms of the presentinvention are eukaryotic microorganisms which are capable of producinglipids at a fermentation medium oxygen level of about less than 3% ofsaturation.

In still another aspect of the present invention, microorganisms aregrown in a fed-batch process. Moreover,

Yet still another aspect of the present invention provides maintainingan oxygen level of less than about 3% of saturation in the fermentationmedium during second half of the fermentation process.

Another embodiment of the present invention provides a process forproducing eukaryotic microbial lipids comprising:

-   -   (a) growing eukaryotic microorganisms in a fermentation medium        to increase the biomass density of said fermentation medium to        at least about 100 g/L;    -   (b) providing a fermentation condition sufficient to allow said        microorganisms to produce said lipids; and    -   (c) recovering said lipids,        wherein greater than about 15% of said lipids are        polyunsaturated lipids.

Another aspect of the present invention provides a lipid recovery stepwhich comprises:

-   -   (d) removing water from said fermentation medium to provide dry        microorganisms; and    -   (e) isolating said lipids from said dry microorganisms.

Preferably, the water removal step comprises contacting the fermentationmedium directly on a drum-dryer without prior centrifugation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table and a plot of various lipid production parameters of amicroorganism versus the amount of dissolved oxygen level in afermentation medium.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for growing microorganisms,such as, for example, fungi (including yeast), algae, and protists.Preferably, microorganisms are selected from the group consisting ofalgae, protists and mixtures thereof. More preferably, microorganismsare algae. Moreover, the process of the present invention can be used toproduce a variety of lipid compounds, in particular unsaturated lipids,preferably polyunsaturated lipids (i.e., lipids containing at least 2unsaturated carbon-carbon bonds, e.g., double bonds), and morepreferably highly unsaturated lipids (i.e., lipids containing 4 or moreunsaturated carbon-carbon bonds) such as omega-3 and/or omega-6polyunsaturated fatty acids, including docosahexaenoic acid (i.e., DHA);and other naturally occurring unsaturated, polyunsaturated and highlyunsaturated compounds. As used herein, the term “lipid” includesphospholipids; free fatty acids; esters of fatty acids;triacylglycerols; sterols and sterol esters; carotenoids; xanthophyls(e.g., oxycarotenoids); hydrocarbons; and other lipids known to one ofordinary skill in the art.

More particularly, processes of the present invention are useful inproducing eukaryotic microbial polyenoic fatty acids, carotenoids,fungal sterols, phytosterols, xanthophyls, ubiquinones, other compoundswhich require oxygen for producing unsaturated carbon-carbon bonds(i.e., aerobic conditions), and secondary metabolites thereof.Specifically, processes of the present invention are useful in growingmicroorganisms which produce polyenoic fatty acid(s) and for producingmicrobial polyenoic fatty acid(s).

While processes of the present invention can be used to grow a widevariety of microorganisms and to obtain polyunsaturated lipid containingcompounds produced by the same, for the sake of brevity, convenience andillustration, this detailed description of the invention will discussprocesses for growing microorganisms which are capable of producinglipids comprising omega-3 and/or omega-6 polyunsaturated fatty acids, inparticular microorganisms which are capable of producing DHA. Moreparticularly, preferred embodiments of the present invention will bediscussed with reference to a process for growing marine microorganisms,in particular algae, such as Thraustochytrids of the orderThraustochytriales, more specifically Thraustochytriales of the genusThraustochytrium and Schizochytrium, including Thraustochytriales whichare disclosed in commonly assigned U.S. Pat. Nos. 5,340,594 and5,340,742, both issued to Barclay, all of which are incorporated hereinby reference in their entirety. It is to be understood, however, thatthe invention as a whole is not intended to be so limited, and that oneskilled in the art will recognize that the concept of the presentinvention will be applicable to other microorganisms producing a varietyof other compounds, including other lipid compositions, in accordancewith the techniques discussed herein.

Assuming a relatively constant production rate of lipids by an algae, itis readily apparent that the higher biomass density will lead to ahigher total amount of lipids being produced per volume. Currentconventional fermentation processes for growing algae yield a biomassdensity of from about 50 to about 80 g/L or less. The present inventorshave found that by using processes of the present invention, asignificantly higher biomass density than currently known biomassdensity can be achieved. Preferably, processes of the present inventionproduces biomass density of at least about 100 g/L, more preferably atleast about 130 g/L, still more preferably at least about 150 g/L, yetstill more preferably at least about 170 g/L, and most preferablygreater than 200 g/L. Thus, with such a high biomass density, even ifthe lipids production rate of algae is decreased slightly, the overalllipids production rate per volume is significantly higher than currentlyknown processes.

Processes of the present invention for growing microorganisms of theorder Thraustochytriales include adding a source of carbon and a sourceof nitrogen to a fermentation medium comprising the microorganisms at arate sufficient to increase the biomass density of the fermentationmedium to those described above. This fermentation process, where asubstrate (e.g., a carbon source and a nitrogen source) is added inincrements, is generally referred to as a fed-batch fermentationprocess. It has been found that when the substrate is added to a batchfermentation process the large amount of carbon source present (e.g.,about 200 g/L or more per 60 g/L of biomass density) had a detrimentaleffect on the microorganisms. Without being bound by any theory, it isbelieved that such a high amount of carbon source causes detrimentaleffects, including osmotic stress, for microorganisms and inhibitsinitial productivity of microorganisms. Processes of the presentinvention avoid this undesired detrimental effect while providing asufficient amount of the substrate to achieve the above describedbiomass density of the microorganisms.

Processes of the present invention for growing microorganisms caninclude a biomass density increasing stage. In the biomass densityincreasing stage, the primary objective of the fermentation process isto increase the biomass density in the fermentation medium to obtain thebiomass density described above. The rate of carbon source addition istypically maintained at a particular level or range which does not causea significant detrimental effect on productivity of microorganisms. Anappropriate range of the amount of carbon source needed for a particularmicroorganism during a fermentation process is well known to one ofordinary skill in the art. Preferably, a carbon source of the presentinvention is a non-alcoholic carbon source, i.e., carbon source thatdoes not contain alcohol. As used herein, an “alcohol” refers to acompound having 4 or less carbon atoms with one hydroxy group, e.g.,methanol, ethanol and isopropanol. More preferably, a carbon source ofthe present invention is a carbohydrate, including, but not limited to,fructose, glucose, sucrose, molasses, and starch. Other suitable simpleand complex carbon sources and nitrogen sources are disclosed in theabove-referenced patents. Typically, however, a carbohydrate, preferablycorn syrup, is used as the primary carbon source.

A particularly preferred nitrogen source is inorganic ammonium salt,more preferably ammonium salts of sulfate, hydroxide, and mostpreferably ammonium hydroxide.

When ammonium is used as a nitrogen source, the fermentation mediumbecomes acidic if it is not controlled by base addition or buffers. Whenammonium hydroxide is used as the primary nitrogen source, it can alsobe used to provide a pH control. The microorganisms of the orderThraustochytriales, in particular Thraustochytriales of the genusThraustochytrium and Schizochytrium, will grow over a wide pH range,e.g., from about pH 5 to about pH 11. A proper pH range for fermentationof a particular microorganism is within the knowledge of one skilled inthe art.

Processes of the present invention for growing microorganisms can alsoinclude a production stage. In this stage, the primary use of thesubstrate by the microorganisms is not increasing the biomass densitybut rather using the substrate to produce lipids. It should beappreciated that lipids are also produced by the microorganisms duringthe biomass density increasing stage; however, as stated above, theprimary goal in the biomass density increasing stage is to increase thebiomass density. Typically, during the production stage the addition ofthe nitrogen substrate is reduced or preferably stopped.

It was previously generally believed that the presence of dissolvedoxygen in the fermentation medium is crucial in the production ofpolyunsaturated compounds by eukaryotic microorganisms including omega-3and/or omega-6 polyunsaturated fatty acids. Thus, a relatively largeamount of dissolved oxygen in the fermentation medium was generallybelieved to be preferred. Surprisingly and unexpectedly, however, thepresent inventors have found that the production rate of lipids isincreased dramatically when the dissolved oxygen level during theproduction stage is reduced. Thus, while the dissolved oxygen level inthe fermentation medium during the biomass density increasing stage isat least about 8% of saturation, and preferably at least about 4% ofsaturation, during the production stage the dissolved oxygen level inthe fermentation medium is reduced to about 3% of saturation or less,preferably about 1% of saturation or less, and more preferably about 0%of saturation. In one particular embodiment of the present invention,the amount of dissolved oxygen level in the fermentation medium isvaried during the fermentation process. For example, for a fermentationprocess with total fermentation time of from about 90 hours to about 100hours, the dissolved oxygen level in the fermentation medium ismaintained at about 8% during the first 24 hours, about 4% from about24^(th) hour to about 40^(th) hour, and about 0.5% or less from about40^(th) hour to the end of the fermentation process.

The amount of dissolved oxygen present in the fermentation medium can becontrolled by controlling the amount of oxygen in the head-space of thefermentor, or preferably by controlling the speed at which thefermentation medium is agitated (or stirred). For example, a highagitation (or stirring) rate results in a relatively higher amount ofdissolved oxygen in the fermentation medium than a low agitation rate.For example, in a fermentor of about 14,000 gallon capacity theagitation rate is set at from about 50 rpm to about 70 rpm during thefirst 12 hours, from about 55 rpm to about 80 rpm during about 121 hourto about 18^(th) hour and from about 70 rpm to about 90 rpm from about18^(th) hour to the end of the fermentation process to achieve thedissolved oxygen level discussed above for a total fermentation processtime of from about 90 hours to about 100 hours. A particular range ofagitation speeds needed to achieve a particular amount of dissolvedoxygen in the fermentation medium can be readily determined by one ofordinary skill in the art.

A preferred temperature for processes of the present invention is atleast about 20° C., more preferably at least about 25° C., and mostpreferably at least about 30° C. It should be appreciated that coldwater can retain a higher amount of dissolved oxygen than warm water.Thus, a higher fermentation medium temperature has additional benefit ofreducing the amount of dissolved oxygen, which is particularly desiredas described above.

Certain microorganisms may require a certain amount of saline mineralsin the fermentation medium. These saline minerals, especially chlorideions, can cause corrosion of the fermentor and other downstreamprocessing equipment. To prevent or reduce these undesired effects dueto a relatively large amount of chloride ions present in thefermentation medium, processes of the present invention can also includeusing non-chloride containing sodium salts, preferably sodium sulfate,in the fermentation medium as a source of saline (i.e., sodium). Moreparticularly, a significant portion of the sodium requirements of thefermentation are supplied as non-chloride containing sodium salts. Forexample, less than about 75% of the sodium in the fermentation medium issupplied as sodium chloride, more preferably less than about 50% andmore preferably less than about 25%. The microorganisms of the presentinvention can be grown at chloride concentrations of less than about 3g/L, more preferably less than about 500 mg/L, more preferably less thanabout 250 mg/L and more preferably between about 60 mg/L and about 120mg/L.

Non-chloride containing sodium salts can include soda ash (a mixture ofsodium carbonate and sodium oxide), sodium carbonate, sodiumbicarbonate, sodium sulfate and mixtures thereof, and preferably includesodium sulfate. Soda ash, sodium carbonate and sodium bicarbonate tendto increase the pH of the fermentation medium, thus requiring controlsteps to maintain the proper pH of the medium. The concentration ofsodium sulfate is effective to meet the salinity requirements of themicroorganisms, preferably the sodium concentration is (expressed as g/Lof Na) at least about 1 g/L, more preferably in the range of from about1 g/L to about 50 g/L and more preferably in the range of from about 2.0g/L to about 25 g/L.

Various fermentation parameters for inoculating, growing and recoveringmicroorganisms are discussed in detail in U.S. Pat. No. 5,130,242, whichis incorporated herein by reference in its entirety. Any currently knownisolation methods can be used to isolate microorganisms from thefermentation medium, including centrifugation, filtration, decantation,and solvent evaporation. It has been found by the present inventors thatbecause of such a high biomass density resulting from processes of thepresent invention, when a centrifuge is used to recover themicroorganisms it is preferred to dilute the fermentation medium byadding water, which reduces the biomass density, thereby allowing moreeffective separation of microorganisms from the fermentation medium.

Preferably, the microorganisms are recovered in a dry form from thefermentation medium by evaporating water from the fermentation medium,for example, by contacting the fermentation medium directly (i.e.,without pre-concentration, for example, by centrifugation) with a dryersuch as a drum-dryer apparatus, i.e., a direct drum-dryer recoveryprocess. When using the direct drum-dryer recovery process to isolatemicroorganisms, typically a steam heated drum-dryer is employed. Inaddition when using the direct drum-dryer recovery process, the biomassdensity of the fermentation medium is preferably at least about 130 g/L,more preferably at least about 150 g/L, and most preferably at leastabout 180 g/L. This high biomass density is generally desired for thedirect drum-dryer recovery process because at a lower biomass density,the fermentation medium comprises a sufficient amount of water to coolthe drum significantly, thus resulting in incomplete drying ofmicroorganisms. Other methods of drying cells, including spray-drying,are well known to one of ordinary skill in the art.

Processes of the present invention provide a lipid production rate of atleast about 0.5 g/L/hr, preferably at least about 0.7 g/L/hr, morepreferably at least about 0.9 g/L/hr, and most preferably at least about1.0 g/L/hr. Moreover, lipids produced by processes of the presentinvention contain polyunsaturated lipids in the amount greater thanabout 15%, preferably greater than about 20%, more preferably greaterthan about 25%, still more preferably greater than about 30%, and mostpreferably greater than about 35%. Lipids can be recovered from eitherdried microorganisms or from the microorganisms in the fermentationmedium. Generally, at least about 20% of the lipids produced by themicroorganisms in the processes of the present invention are omega-3and/or omega-6 polyunsaturated fatty acids, preferably at least about30% of the lipids are omega-3 and/or omega-6 polyunsaturated fattyacids, more preferably at least about 40% of the lipids are omega-3and/or omega-6 polyunsaturated fatty acids, and most preferably at leastabout 50% of the lipids are omega-3 and/or omega-6 polyunsaturated fattyacids. Alternatively, processes of the present invention provides a DHAproduction rate of at least about 0.2 g of DHA/L/hr, preferably at leastabout 0.3 g of DHA/L/hr, more preferably at least about 0.4 g ofDHA/L/hr, and most preferably at least about 0.5 g of DHA/L/hr. Stillalternatively, at least about 25% of the lipid is DHA (based on totalfatty acid methyl ester), preferably at least about 30%, more preferablyat least about 35%, and most preferably at least about 40%.

Microorganisms, lipids extracted therefrom, the biomass remaining afterlipid extraction or combinations thereof can be used directly as a foodingredient, such as an ingredient in beverages, sauces, dairy basedfoods (such as milk, yogurt, cheese and ice-cream) and baked goods;nutritional supplement (in capsule or tablet forms); feed or feedsupplement for any animal whose meat or products are consumed by humans;food supplement, including baby food and infant formula; andpharmaceuticals (in direct or adjunct therapy application). The term“animal” means any organism belonging to the kingdom Animalia andincludes, without limitation, any animal from which poultry meat,seafood, beef, pork or lamb is derived. Seafood is derived from, withoutlimitation, fish, shrimp and shellfish. The term “products” includes anyproduct other than meat derived from such animals, including, withoutlimitation, eggs, milk or other products. When fed to such animals,polyunsaturated lipids can be incorporated into the flesh, milk, eggs orother products of such animals to increase their content of theselipids.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES

The strain of Schizochytrium used in these examples produces two primarypolyenoic acids, DHAn-3 and DPAn-6 in the ratio of generally about 3:1,and small amounts of other polyenoic acids, such as EPA and C20:3, undera wide variety of fermentation conditions. Thus, while followingexamples only list the amount of DHA, one can readily calculate theamount of DPA produced by using the above disclosed ratio.

Example 1

This example illustrates the affect of oxygen content in a fermentationmedium on lipid productivity.

Fermentation results of Schizochytrium at various levels of dissolvedoxygen content were measured. The results are shown in FIG. 1, where RCSis residual concentration of sugar, and DCW is dry-cell weight.

Example 2

This example illustrates the reproducibility of processes of the presentinvention.

Microorganisms were produced using fermentors with a nominal workingvolume of 1,200 gallons. The resulting fermentation broth wasconcentrated and microorganisms were dried using a drum-dryer. Lipidsfrom aliquots of the resulting microorganisms were extracted andpurified to produce a refined, bleached, and deodorized oil.Approximately 3,000 ppm of d-1-α-tocopheryl acetate was added fornutritional supplementation purposes prior to analysis of the lipid.

Nine fermentations of Schizochytrium were run and the results are shownin Table 1. The dissolved oxygen level was about 8% during the first 24hours and about 4% thereafter.

TABLE 1 Fed-batch fermentation results for the production of DHA. AgeYield¹ DHA FAME² Entry (Hrs) (g/L) (%) (%) Productivity³ 1 100.3 160.717.8 49.5 0.285 2 99.8 172.4 19.4 51.3 0.335 3 84.7 148.7 14.4 41.40.253 4 90.2 169.5 19.7 53.9 0.370 5 99.0 164.1 12.5 38.9 0.207 6 113.0187.1 19.7 47.2 0.326 7 97.0 153.5 13.7 41.0 0.217 8 92.8 174.8 16.448.6 0.309 Avg⁴ 97.1 166.4 16.7 46.5 0.288 Std⁵ 8.4 12.3 2.9 5.4 0.058CV⁶(%) 8.7 7.4 17.3 11.7 20.2 ¹actual yield of biomass density. ²totalfatty acid methyl esters. ³(grams of DHA)/L/Hr. ⁴average. ⁵standarddeviation. ⁶coefficients of variability. Coefficients of variabilityvalues below 5% indicates a process which has excellent reproducibility,values between 5% and 10% indicates a process which has goodreproducibility and values between 10% and 20% indicates a process whichhas reasonable reproducibility.

Corn syrup was fed until the volume in the fermentor reached about 1,200gallons, at which time the corn syrup addition was stopped. Thefermentation process was stopped once the residual sugar concentrationfell below 5 g/L. The typical age, from inoculation to final, was about100 hours.

The fermentation broth, i.e., fermentation medium, was diluted withwater using approximately a 2:1 ratio to reduce the ash content of thefinal product and help improve phase separation during thecentrifugation step. The concentrated cell paste was heated to 160° F.(about 71° C.) and dried on a Blaw Knox double-drum dryer (42″×36″).Preferably, however, microorganisms are dried directly on a drum-dryerwithout prior centrifugation.

The analysis result of lipids extracted from aliquots of each entries inTable 1 is summarized in Table 2.

TABLE 2 Analysis of lipids from microorganisms of Table 1. % DHArelative Total Lipid % Entry to FAME¹ by wt. 1 36.0 72.3 2 37.8 70.3 334.8 61.5 4 36.5 74.8 5 32.1 52.8 6 41.7 67.7 7 33.4 49.9 8 33.7 61.4Avg 35.8 63.8 Std.³ 3.0 9.1 CV⁴ (%) 8.5 14.2 ¹see Table 1 ²seediscussion above ³standard deviation ⁴coefficients of variabilityCoefficients of variability values below 5% indicates a process whichhas excellent reproducibility, values between 5% and 10% indicates aprocess which has good reproducibility and values between 10% and 20%indicates a process which has reasonable reproducibility.

Unless otherwise stated, the fermentation medium used throughout theExamples section includes the following ingredients, where the firstnumber indicates nominal target concentration and the number inparenthesis indicates acceptable range: sodium sulfate 12 g/L (11-13);KCl 0.5 g/L (0.45-0.55); MgSO₄.7H₂O 2 g/L (1.8-2.2); Hodag K-60 antifoam0.35 g/L (0.3-0.4); K₂SO₄ 0.65 g/L (0.60-0.70); KH₂PO₄ 1 g/L (0.9-1.1);(NH₄)₂SO₄ 1 g/L (0.95-1.1); CaCl₂.2H₂O 0.17 g/L (0.15-0.19); 95 DE cornsyrup (solids basis) 4.5 g/L (2-10); MnCl₂.4H₂O 3 mg/L (2.7-3.3);ZnSO₄.7H₂O 3 mg/L (2.7-3.3); CoCl₂.6H₂O 0.04 mg/L (0.035-0.045);Na₂MoO₄.2H₂O 0.04 mg/L (0-0.045); CuSO₄.5H₂O 2 mg/L (1.8-2.2);NiSO₄.6H₂O 2 mg/L (1.8-2.2); FeSO₄.7H₂O 10 mg/L (9-11); thiamine 9.5mg/L (4-15); vitamin B₁₂ 0.15 mg/L (0.05-0.25) and Ca_(1/2) Pantothenate3.2 mg/L (1.3-5.1). In addition, 28% NH₄OH solution is used as thenitrogen source.

The ash content of the dried microorganisms is about 6% by weight.

Example 3

This example illustrates the effect of reduced dissolved oxygen level inthe fermentation medium on the productivity of microorganisms usingG-tank scale.

Using the procedure described in Example 2, a 14,000 gallon nominalvolume fermentation was conducted using Schizochytrium, which can beobtained using isolation processes disclosed in the above mentioned U.S.Pat. Nos. 5,340,594 and 5,340,742. The dissolved oxygen level in thefermentation medium was about 8% during the first 24 hours, about 4%from the 24^(th) hour to the 40^(th) hour and about 0.5% from the40^(th) hour to the end of fermentation process. Results of this lowerdissolved oxygen level in fermentation medium processes are shown inTable 3.

TABLE 3 14,000 gallon scale fermentation of Schizochytrium. % DHA DHAAge Yield % % rel. to Productivity Entry (Hrs) (g/L) DHA FAME FAME (g ofDHA/L/hr) 1 82.0 179.3 21.7 52.4 41.4 0.474 2 99.0 183.1 22.3 55.0 40.50.412 3 72.0 159.3 — — 40.9 — 4 77.0 161.3 — — 43.2 — 5 100.0 173.0 23.953.3 44.9 0.413 6 102.0 183.3 21.6 50.8 42.6 0.388 7 104.0 185.1 23.755.0 43.1 0.422 8 88.0 179.3 22.3 52.6 42.4 0.454 9 100.0 166.4 22.553.5 42.1 0.374 10 97.0 182.6 22.8 51.6 44.1 0.429 11 87.5 176.5 19.845.6 43.5 0.399 12 67.0 170.8 18.8 48.1 39.1 0.479 13 97.0 184.9 23.252.7 44.0 0.442 14 102.0 181.9 23.6 52.9 44.6 0.421 15 102.0 186.9 19.947.8 41.8 0.365 16 97.0 184.4 19.6 45.5 43.0 0.373 17 98.0 174.7 19.745.1 43.7 0.351 18 103.5 178.8 18.3 44.5 41.2 0.316 19 102.0 173.7 15.843.1 36.7 0.269 20 94.0 190.4 19.3 46.9 41.1 0.391 21 72.0 172.5 22.852.8 43.2 0.546 22 75.0 173.1 21.0 51.7 40.8 0.485 23 75.0 152.7 20.350.3 40.4 0.413 24 75.5 172.5 21.9 51.7 42.3 0.500 25 61.0 156.4 17.345.7 37.8 0.444 26 74.5 150.6 20.2 50.1 40.2 0.408 27 70.5 134.3 14.840.6 36.6 0.282 28 75.5 146.1 21.3 49.7 42.8 0.412 29 82.0 174.3 21.450.4 42.5 0.455 30 105.0 182.3 21.7 50.7 42.8 0.377 31 66.0 146.2 16.444.6 36.7 0.363 Avg 87.2 171.5 20.6 49.5 41.6 0.409 Std 13.9 14.1 2.43.8 2.3 0.061 CV 16.0% 8.2% 11.6% 7.7% 5.5% 15.0%

Example 4

This example illustrates the effect of reduced dissolved oxygen level inthe fermentation medium on the productivity of microorganisms on a41,000 gallon scale.

Same procedure as Example 3 in a 41,000 gallon fermentor was performed.Results are shown in Table 4.

TABLE 4 41,000 gallon scale fermentation of Schizochytrium % DHA DHA AgeYield % % rel. to Productivity Entry (Hrs) (g/L) DHA FAME FAME (g ofDHA/L/hr) 1 75.0 116.1 17.3 46.1 37.4 0.268 2 99.0 159.3 17.4 47.0 37.10.280 3 103.0 152.6 16.0 47.2 33.8 0.237 4 68.0 136.8 17.9 45.9 39.10.360 5 84.0 142.0 17.5 47.0 37.2 0.296 Avg 85.8 141.4 17.2 46.6 36.90.288 Std 15.1 16.6 0.7 0.6 1.9 0.046 CV 17.5% 11.8 4.2% 1.3% 5.2% 15.8%

Example 5

This example illustrates the affect of extra nitrogen on thefermentation process of the present invention.

Four sets of 250-L scale fed-batch experiments were conducted using aprocedure similar to Example 3. Two control experiments and twoexperiments containing extra ammonia (1.15× and 1.25× the normal amount)were conducted. Results are shown in Table 5.

TABLE 5 Affects of extra ammonia on fermentation of Schizochytrium. AgeYield Biomass Conversion DHA FAME DHA (hrs) (g/L) ProductivityEfficiency Content Content Productivity Sugar target: 7 g/L, Base pH setpoint: 5.5, Acid pH set point: 7.3, 1.0× NH₃ 48 178 3.71 g/L/hr 51.5%10.7% 37.8% 0.40 g/L/hr 60 185 3.08 g/L/hr 46.9% 16.3% 47.2% 0.50 g/L/hr72 205 2.85 g/L/hr 45.2% 17.4% 47.4% 0.50 g/L/hr 84 219 2.61 g/L/hr43.8% 17.1% 45.5% 0.45 g/L/hr 90 221 2.46 g/L/hr 44.1% 18.4% 48.9% 0.45g/L/hr Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point:7.3, 1.15× NH₃ 48 171 3.56 g/L/hr 55.6% 12.0% 36.3% 0.43 g/L/hr 60 1973.28 g/L/hr 54.6% 9.4% 38.4% 0.31 g/L/hr 72 191 2.65 g/L/hr 52.8% 9.4%40.0% 0.25 g/L/hr 84 190 2.26 g/L/hr 52.5% 10.0% 42.5% 0.23 g/L/hr 90189 2.10 g/L/hr 52.2% 9.2% 43.3% 0.19 g/L/hr Sugar target: 7 g/L, BasepH set point: 5.5, Acid pH set point: 7.3, 1.25× NH₃ 48 178 3.71 g/L/hr56.4% 11.5% 33.7% 0.43 g/L/hr 60 179 2.98 g/L/hr 48.6% 10.3% 36.0% 0.31g/L/hr 72 180 2.50 g/L/hr 48.8% 12.0% 37.6% 0.30 g/L/hr 84 181 2.15g/L/hr 46.1% 13.6% 40.1% 0.29 g/L/hr 90 185 2.06 g/L/hr 45.7% 12.6%40.7% 0.26 g/L/hr Sugar target: 7 g/L, Base pH set point: 5.5, Acid pHset point: 7.3, 1.0× NH₃ 48 158 3.29 g/L/hr 55.7% 13.1% 36.5% 0.43g/L/hr 60 174 2.90 g/L/hr 48.9% 17.9% 39.2% 0.52 g/L/hr 72 189 2.63g/L/hr 45.7% 21.0% 39.4% 0.55 g/L/hr 84 196 2.33 g/L/hr 44.1% 22.4 40.1%0.52 g/L/hr 90 206 2.29 g/L/hr 44.8% 22.1% 40.3% 0.51 g/L/hrIn general, extra nitrogen has a negative effect on fermentationperformance, as significant reductions were observed in the DHAproductivity for the two batches where extra ammonia were added. Asshown on Table 5, the control batches resulted in final DHA levels of18.4% and 22.1% versus the 9.2% (1.15× ammonia) and 12.6% (1.25×ammonia) for extra nitrogen supplemented batches.

Example 6

This example shows a kinetic profile of a fermentation process of thepresent invention.

A 1000 gallon scale fed-batch experiment was conducted using a proceduresimilar to Example 3. Kinetic profile of the fermentation process isshown in Table 6.

TABLE 6 Kinetic Profile for a 1,000 gallon scale Fed-Batch fermentationof Schizochytrium. Age Yield Biomass Conversion % DHA % FAME DHA (hrs)(g/L) Productivity Efficiency Content Content Productivity 24 118 4.92g/L/hr 78.2% 7.4 18.8 0.36 g/L/hr 30 138 4.60 g/L/hr 60.3% 10.6 30.90.49 g/L/hr 36 138 3.83 g/L/hr 46.6% 11.6 36.5 0.44 g/L/hr 42 175 4.17g/L/hr 49.8% 13.4 41.7 0.56 g/L/hr 48 178 3.71 g/L/hr 45.1% 18.7 52.80.69 g/L/hr  48* 164 3.42 g/L/hr 41.5% 15.3 33.1 0.52 g/L/hr 54 196 3.63g/L/hr 45.7% 16.6 51.2 0.60 g/L/hr 60 190 3.17 g/L/hr 41.7% 16.9 33.90.54 g/L/hr 72 189 2.62 g/L/hr 39.1% 15.6 31.8 0.41 g/L/hr 84 195 2.32g/L/hr 38.5% 16.4 32.7 0.38 g/L/hr 90 200 2.22 g/L/hr 39.0% 18.8 33.30.42 g/L/hr 90 171 1.90 g/L/hr 33.3% 22.2 61.6  0.42 g/L/hr** *Twoseparate samples were analyzed at 48 hrs. **This is for a washeddry-cell weights (DCW) sample. Other reported values are for unwashedsamples.

Example 7

This example illustrates affect of the amount of carbon source onproductivity.

Three different fermentation processed using the process of Example 3were conducted using various amounts of carbon source. Results are shownon Table 7.

TABLE 7 Fermentation results for various amounts of carbon source onfermentation of Schizochytrium. Age Yield Carbon Conversion % DHA % FAMEProductivity (hrs) (g/L) Charge Efficiency Content Content (g/L/hr) 90171 51.3% 33.3% 22.2 61.6 0.42 94 122 40.5% 30.1% 19.1 57.3 0.25 59 7320.0% 36.5% 11.9 40.8 0.15

The present invention, in various embodiments, includes components,methods, processes, systems and/or apparatus substantially as depictedand described herein, including various embodiments, subcombinations,and subsets thereof. Those of skill in the art will understand how tomake and use the present invention after understanding the presentdisclosure. The present invention, in various embodiments, includesproviding devices and processes in the absence of items not depictedand/or described herein or in various embodiments hereof, including inthe absence of such items as may have been used in previous devices orprocesses, e.g., for improving performance, achieving ease and\orreducing cost of implementation.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1. A process for growing microorganisms of the order Thraustochytrialesby conducting a fermentation of the microorganisms in a fermentationmedium comprising a carbon source and a limiting nutrient source, theprocess comprising: a) providing the fermentation medium comprising thecarbon source and limiting nutrient source; b) adding the microorganismsto the fermentation medium; c) adding an additional amount of the carbonsource and limiting nutrient source to the fermentation medium toincrease the biomass density of the fermentation medium when thedissolved oxygen content of the fermentation medium is at least 4% ofsaturation; d) adding an additional amount of the carbon source whilelimiting the amount of limiting nutrient source added to thefermentation medium and allowing the dissolved oxygen content of thefermentation medium to decrease to less than about 3% of saturation;wherein lipid production is induced in the microorganisms; and e)recovering at least some of the lipids from the microorganisms; whereinthe process produces at least an average rate of 0.5 grams lipids perliter of fermentation medium per hour fermentation wherein at leastabout 15% of the total lipids produced by the microorganisms ispolyunsaturated fatty acids, and wherein the lipids are at least 20% byweight of the dry cell weight of the biomass.
 2. The process of claim 1,wherein the process is a fed batch process.
 3. The process of claim 1,wherein the microorganisms produce lipids containing polyunsaturatedfatty acids when the dissolved oxygen content of the fermentation mediumis less than 1% of saturation.
 4. The process of claim 1, wherein themicroorganisms are selected from the group consisting ofThraustochytrium, Schizochytrium, and mixtures thereof.
 5. The processof claim 1, wherein the process produces on average at least 0.2 gramsdocosahexaenoic acid per liter of fermentation medium per hourfermentation.
 6. The process of claim 1, wherein the microorganisms areSchizochytrium.